WO2023081905A2 - Plateforme de détection pour oligonucléotides non marqués - Google Patents

Plateforme de détection pour oligonucléotides non marqués Download PDF

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WO2023081905A2
WO2023081905A2 PCT/US2022/079434 US2022079434W WO2023081905A2 WO 2023081905 A2 WO2023081905 A2 WO 2023081905A2 US 2022079434 W US2022079434 W US 2022079434W WO 2023081905 A2 WO2023081905 A2 WO 2023081905A2
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strand
dsna
dsna molecule
molecule
bound
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PCT/US2022/079434
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WO2023081905A3 (fr
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Valeria Tohver MILAM
Mary Catherine Adams
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Georgia Tech Research Corporation
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Publication of WO2023081905A3 publication Critical patent/WO2023081905A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the various embodiments of the present disclosure relate generally to systems and methods for making and using nucleic acid detection platforms, and more particularly to nucleic acid detection platforms free from expensive PCR instrumentation or methods requiring multi-step manufacturing steps.
  • Detection platforms that use more readily available equipment and reagents such as molecular beacons (i.e., self-folded hairpins) and double-stranded nucleic acid probes (dsprobes) typically involve fluorescence spectroscopy of oligonucleotide solutions initially in a quenched or signal-off state.
  • molecular beacons i.e., self-folded hairpins
  • dsprobes double-stranded nucleic acid probes
  • dsprobes double-stranded nucleic acid probes
  • the present disclosure relates to systems and methods for making and using nucleic acid detection platforms.
  • An exemplary embodiment of the present disclosure provides a composition comprising microspheres and at least one double-stranded nucleic acid (dsNA) molecule.
  • One strand of the at least one dsNA molecule can be bound to an outer surface of the microspheres at a 3 ’end.
  • the bound strand of the at least one dsNA molecule can include a recognition domain.
  • the bound strand can also contain a quenchable colorimetric indicator at a 5’ end.
  • the other strand of the at least one dsNA molecule can be soluble and can contain a quencher of the colorimetric indicator at a 3 ’ end.
  • the microspheres can be coated in streptavidin and the bound strand of the at least one dsNA molecule comprises biotin at the 3 ’ end.
  • the bound strand of the at least one dsNA molecule further can include a toehold domain at the 3’ end.
  • the toehold domain ccan include between 1 and 12 nucleotides.
  • the quenchable colorimetric indicator can be a fluorescent indicator.
  • the soluble strand of the at least one dsNA molecule can be configured to be displaced by a single stranded NA molecule, thereby displacing the quencher of the colorimetric indicator such that the colorimetric indicator is active.
  • the active colorimetric indicator can be detectable and/or quantifiable.
  • the dsNA molecule can include DNA, RNA, or LNA.
  • the bound strand of the at least one dsNA molecule can include a COVID- 19-specific nucleic acid, or encodes a COVID-19-specific protein.
  • the bound strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 1, 12, and 21.
  • the soluble strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 2-11, 13-20, and 22.
  • kits for detecting the presence of a nucleic acid in a sample can include microspheres and at least one double-stranded nucleic acid (dsNA) molecule.
  • dsNA double-stranded nucleic acid
  • One strand of the at least one dsNA molecule can be bound to an outer surface of the microspheres at a 3 ’end.
  • the bound strand of the at least one dsNA molecule can include a recognition domain.
  • the bound strand can also contain a quenchable colorimetric indicator at a 5’ end.
  • the other strand of the at least one dsNA molecule can be soluble and can contain a quencher of the colorimetric indicator at a 3’ end.
  • the microspheres can be coated in streptavidin and the bound strand of the at least one dsNA molecule comprises biotin at the 3 ’ end.
  • the bound strand of the at least one dsNA molecule further can include a toehold domain at the 3 ’ end.
  • the toehold domain can include between 1 and 12 nucleotides.
  • the quenchable colorimetric indicator can be a fluorescent indicator.
  • the soluble strand of the at least one dsNA molecule can be configured to be displaced by a single stranded NA molecule, thereby displacing the quencher of the colorimetric indicator such that the colorimetric indicator is active.
  • the active colorimetric indicator can be detectable and/or quantifiable.
  • the dsNA molecule can include DNA, RNA, or LNA.
  • the bound strand of the at least one dsNA molecule can include a COVID- 19-specific nucleic acid, or encodes a COVID-19-specific protein.
  • the bound strand of the at least one dsNA molecule can include a sequence selected from the group consisting of SEQ ID NOs: 1, 12, and 21.
  • the soluble strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 2-11, 13-20, and 22.
  • the sample can include a biological sample, a water sample, a wastewater sample, an environmental sample, a food sample, and/or a food contact surface sample.
  • the biological sample can include tissue, blood, serum, plasma, saliva, nasopharyngeal swab or secretion, urine, cerebrospinal fluid, lymphatic fluid, sputum, cell lysate, and the like.
  • An exemplary embodiment of the present disclosure provides a method of detecting and/or quantifying a nucleic acid.
  • the method can include incubating the nucleic acid with a composition and measuring the colorimetric indicator to detect and/or quantify the nucleic acid.
  • the composition can include microspheres and at least one double-stranded nucleic acid (dsNA) molecule.
  • dsNA double-stranded nucleic acid
  • One strand of the at least one dsNA molecule can be bound to an outer surface of the microspheres at a 3 ’end.
  • the bound strand of the at least one dsNA molecule can include a recognition domain.
  • the bound strand can also contain a quenchable colorimetric indicator at a 5’ end.
  • the other strand of the at least one dsNA molecule can be soluble and can contain a quencher of the colorimetric indicator at a 3 ’ end.
  • the measuring step can be performed by a high-throughput spectrophotometer, a fluorescence activated cell sorting (FACS) system, or a color activated cell sorting system.
  • FACS fluorescence activated cell sorting
  • the microspheres can be coated in streptavidin and the bound strand of the at least one dsNA molecule comprises biotin at the 3 ’ end.
  • the bound strand of the at least one dsNA molecule further can include a toehold domain at the 3 ’ end.
  • the toehold domain can include between 1 and 12 nucleotides.
  • the quenchable colorimetric indicator can be a fluorescent indicator.
  • the dsNA molecule can include DNA, RNA, or LNA.
  • the bound strand of the at least one dsNA molecule can include a COVID- 19-specific nucleic acid, or encodes a COVID-19-specific protein.
  • the bound strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 1, 12, and 21.
  • the soluble strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 2-11, 13-20, and 22.
  • An exemplary embodiment of the present disclosure provides a method of detecting a nucleic acid in a biological sample from a patient.
  • the method can include isolating the nucleic acid from the biological sample, incubating the nucleic acid with a composition, and measuring the colorimetric indicator to detect and/or quantify the nucleic acid.
  • the composition can include microspheres and at least one double-stranded nucleic acid (dsNA) molecule.
  • One strand of the at least one dsNA molecule can be bound to an outer surface of the microspheres at a 3 ’end.
  • the bound strand of the at least one dsNA molecule can include a recognition domain.
  • the bound strand can also contain a quenchable colorimetric indicator at a 5’ end.
  • the other strand of the at least one dsNA molecule can be soluble and can contain a quencher of the colorimetric indicator at a 3 ’ end.
  • the measuring step can be performed by a high-throughput spectrophotometer, a fluorescence activated cell sorting (FACS) system, or a color activated cell sorting system.
  • FACS fluorescence activated cell sorting
  • the microspheres can be coated in streptavidin and the bound strand of the at least one dsNA molecule comprises biotin at the 3 ’ end.
  • the bound strand of the at least one dsNA molecule further can include a toehold domain at the 3 ’ end.
  • the toehold domain can include between 1 and 12 nucleotides.
  • the quenchable colorimetric indicator can be a fluorescent indicator.
  • the dsNA molecule can include DNA, RNA, or LNA.
  • the bound strand of the at least one dsNA molecule can include a COVID- 19-specific nucleic acid, or encodes a COVID-19-specific protein.
  • the bound strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 1, 12, and 21.
  • the soluble strand of the at least one dsNA molecule can have a sequence selected from the group consisting of SEQ ID NOs: 2-11, 13-20, and 22.
  • the patient can be a mammal.
  • the patient can be a human or veterinary animal.
  • the biological sample can include tissue, blood, serum, plasma, saliva, nasopharyngeal swab or secretion, urine, cerebrospinal fluid, lymphatic fluid, sputum, cell lysate, and the like.
  • FIG. 1 provides a schematic of an example of a responsive nucleic acid detection platform, in accordance with an exemplary embodiment of the present invention.
  • FIG. 2 provides a schematic of an example of an unresponsive nucleic acid detection platform, in accordance with an exemplary embodiment of the present invention.
  • FIG. 3 provides a schematic of an example nucleic acid detection platform with initial fluorescent signals, in accordance with an exemplary embodiment of the present invention.
  • FIG. 4 provides a bar graph of molecules of equivalent soluble fluorochrome (MESF) for example nucleic acid detection platforms with addition of various sequences, in accordance with an exemplary embodiment of the present invention.
  • EMF equivalent soluble fluorochrome
  • FIG. 5 provides a bar graph of molecules of equivalent soluble fluorochrome (MESF) for example nucleic acid detection platforms with addition of various sequences, in accordance with an exemplary embodiment of the present invention.
  • EMF equivalent soluble fluorochrome
  • FIG. 6 provides a bar graph of molecules of equivalent soluble fluorochrome (MESF) for an example nucleic acid detection platform with addition of various sequences, in accordance with an exemplary embodiment of the present invention.
  • EMF equivalent soluble fluorochrome
  • FIG. 7 provides a flow-chart of an example method of making nucleic acid detection platforms, in accordance with an exemplary embodiment of the present invention.
  • the term “and/or” may mean “and,” it may mean “or,” it may mean “exclusive-or,” it may mean “one,” it may mean “some, but not all,” it may mean “neither,” and/or it may mean “both.”
  • the term “or” is intended to mean an inclusive “or.”
  • references to “one embodiment,” “an embodiment,” “example embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc. indicate that the embodiment(s) of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may. [0063] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention.
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the term “subject” or “patient” refers to mammals and includes, without limitation, human and veterinary animals. In a preferred embodiment, the subject is human.
  • nucleic acid detection platforms involving double-stranded (ds) probes comprised of quencher-dye sequence pairs exhibit advantages over single-stranded probes including superior target sequence specificity and no prerequisite target labeling.
  • dsprobe design can include finding optimal sequence combinations that balance stability requirements while minimizing spontaneous dsprobe dissociation events (i.e., maintain signal-off state in target absence) with fast, accurate response to a specific target sequence (i.e., selectively trigger signal-on state in target presence).
  • flow cytometry can be used to rapidly interrogate the stability and selective responsiveness of locked nucleic acid (LNA; i.e., a nucleic acid comprising one or more locked nucleotides), RNA dsprobes, and DNA dsprobes to a segment of a pathogen.
  • LNA locked nucleic acid
  • RNA dsprobes i.e., a nucleic acid comprising one or more locked nucleotides
  • DNA dsprobes i.e., a nucleic acid comprising one or more locked nucleotides
  • DNA dsprobes can be detected using the system described herein.
  • a DNA dsprobe with a 15 base-long hybridization partner containing a central abasic site can exhibit very stable, and selective detection of a pathogen such as SARS-CoV-2 RNA.
  • an exemplary embodiment of the present invention provides an example composition 100 of a nucleic acid detection platform in an initially signal-off state.
  • Composition 100 can undergo displacement of one of the sequences by a target sequence 142 to induce a signal from the platform, as indicated in a shift in fluorescence intensity in the plot to the right.
  • Composition 100 can include microspheres 110 or nanobeads functionalized with double-stranded nucleic acid (dsNA) molecules 120.
  • dsNA double-stranded nucleic acid
  • the dsNA molecule can be dsRNA or dsDNA.
  • the nucleic acid molecules can include one or more locked nucleotides to form a locked nucleic acid (“LNA”).
  • LNA locked nucleic acid
  • a dsNA probe can include a ratio of approximately 33% LNA to 67% DNA, although lower amounts of LNA are possible (e.g., 32% LNA to 68% DNA; 30% LNA to 70% DNA; 25% LNA to 75% DNA; 20% LNA to 80% DNA; 15% LNA to 85% DNA; 10% LNA to 90% DNA; 5% LNA to 95% DNA, and any ratio in between, e.g., 28.4% LNA to 71 .6% DNA).
  • Microparticles, or microspheres 110 can be nonfluorescent or fluorescently-labeled and useful in flow cytometry, confocal laser scanning microscopy, light scattering measurements, or particle dynamic analysis.
  • Microspheres 110 can be made with polystyrene or melamine resin, modified with surface functional groups (e.g., methylol groups, amino groups, carboxylate groups, streptavidin, and the like).
  • surface functional groups e.g., methylol groups, amino groups, carboxylate groups, streptavidin, and the like.
  • microspheres can be prepared with variations in chemical composition, size, type of fluorochrome (optional), and surface functional groups.
  • Microparticles can range from about 0.1 pm to about 100 pm (e.g., from about 0.1 pm to about 1 pm, from about 1 pm to about 3 pm, from about 2 pm to about 4 pm, from about 3 pm to about 5 pm, from about 4 pm to about 6 pm, from about 5 pm to about 7 pm, from about 6 pm to about 8 pm, from about 7 pm to about 9 pm, from about 8 pm to about 10 pm, from about 10 pm to about 30 pm, from about 20 pm to about 40 pm, from about 30 pm to about 50 pm, from about 40 pm to about 60 pm, from about 50 pm to about 70 pm, from about 60 pm to about 80 pm, from about 70 pm to about 90 pm, from about 80 pm to about 100 pm, and any size in between, e.g., from about 0.86 pm to about 37 pm).
  • Suitable fluorochromes can include FITC, green fluorescence, rhodamine B, orange fluorescence, nile blue A, red fluorescence, and the like.
  • microspheres 110 can be used to provide consistent instrument performance with minimal data variation for alignment, size calibration, and sorting set-up; convenience to save sample and set compensation for anti-body, reagent, and fluorescent proteins or nucleic acids; and comparable data between samples and instruments by acting as a standard.
  • composition 100 may include nanobeads that can offer intense and stable fluorescent signals.
  • polyacrylnitrile, polystyrene, or PD nanoparticles can be fluorescently labeled and can be less than about 50 nm in diameter (e.g., less than 45 nm, less than a 40 nm, less than 35 nm, less than a 30 nm, less than 25 nm, less than a 20 nm, less than 15 nm, less than a 10 nm, less than 5 nm, and any size in between, e.g., less than about 16.2 nm).
  • microsphere 110 can have a surface 112 functionalized such that one strand 122 of the dsNA molecule 120 can be bound at a 3’ end of strand 122.
  • the dsNA molecule can include DNA, RNA, or locked nucleic acid (LNA).
  • microsphere surface 112 is functionalized with streptavidin to form a strong, but noncovalent bond with strand 122 with a biotin at the 3’ end. Bound strand
  • recognition domain 124 can make up approximately 20% to approximately 99% of the sequence of bound strand 122 (e.g., approximately 22%, approximately 24%, approximately 26%, approximately 28%, approximately 30%, approximately 32%, approximately 34%, approximately 36%, approximately 38%, approximately 40%, approximately 42%, approximately 44%, approximately 46%, approximately 48%, approximately 50%, approximately 52%, approximately 54%, approximately 56%, approximately 58%, approximately 60%, approximately 62%, approximately 64%, approximately 66%, approximately 68%, approximately 70%, approximately 72%, approximately 74%, approximately 76%, approximately 78%, approximately 80%, approximately 82%, approximately 84%, approximately 86%, approximately 88%, approximately 90%, approximately 92%, approximately 94%, approximately 96%, approximately 98%, approximately 99%, or any range in between, e.g., approximately 97.5%).
  • the recognition domain comprises at least 50% nucleic
  • Bound strand 122 can also include a toehold domain 128.
  • Toehold domain 128 can be positioned nearer the 3 ’ end of all bound strands such that a target may hybridize with the bound strands in a similar way.
  • toehold domain 128 can be positioned near the 3’ end of one bound strand, and further from the 3’ end of a neighboring bound strand such that a target may hybridize with the bound strand in a staggered manner along microsphere.
  • the toehold domain 128 may include from about 1 nucleotide to about 12 nucleotides (e.g., 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides).
  • the toehold domain comprises at least 6 nucleotides.
  • Bound strand 122 may also include a quenchable colorimetric indicator 126 at the 5’ end.
  • Quenchable colorimetric indicator 126 can include a fluorescent indicator that may be detected using a suitable fluorescence detection method such as flow cytometry, fluorescence-activated cell sorting, FRET, and the like.
  • quenchable colorimetric indicator 126 can include a dye or other visible color indicator that can be detected using a suitable colorimetric detector such as a spectrophotometer or a color activated cell sorting system.
  • suitable colorimetric indicators may include FAM, Tet M, Tet O, HEX, Cy3, TAMRA, ATTO, Cy5, and the like.
  • bound strand 122 may include from about 1 nucleotide to about 30 nucleotides (e.g., 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides).
  • bound strand 122 may include from about 1 nucleo
  • bound strand 122 may include one or more locked nucleotides.
  • bound strand 122 includes approximately 33% locked nucleotides.
  • the locked nucleotides may be located at every 3rd base starting with the 3rd nucleotide from the immobilized end.
  • bound strand 122 may include one or more abasic sites. In a preferred embodiment, bound strand 122 includes up to 20% abasic sites. The abasic sites may be located in the middle of the strand or may be located along the recognition domain 124 or the toehold domain 128.
  • the soluble strand 132 of dsNA molecule 120 can contain a quencher 136 positioned at the 3’ end of the other strand 132.
  • quencher 136 can specifically quench colorimetric indicator 126 of bound strand 122 such as, for example, Black Hole and Iowa Black quenchers.
  • soluble strand 132 may include from about 1 nucleotide to about 100 nucleotides (e.g., 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 35 nucleotides (e.g., 1 nu
  • soluble strand 132 may include 24 nucleotides.
  • the dsNA probes are specific to a virus or bacterium.
  • viruses and bacteria include pathogenic viruses and bacteria, such as viruses and bacteria that are human pathogens.
  • systems and methods described herein may be used to detect helical and envelope viruses in which RNA can be extracted into a sample.
  • the virus can be SARS-COVID-19.
  • the dsNA probes are specific for environmental contaminants (e.g., contaminants of water, wastewater, or water supplies), food contact surfaces, or food preparation contact surfaces, high contact surfaces such as door handles.
  • any of the above embodiments of the dsNA probes may be used in any of the methods and/or kits described herein.
  • kits can include a microsphere as described herein and dsNA probes as described herein.
  • the dsNA probes can optionally be already bound to the microsphere surface.
  • the kit can be used to test for the presence of a certain nucleic acid in a sample.
  • samples that can be tested with kits according to the invention include biological samples, water samples, wastewater samples, environmental samples, food samples, and/or food contact surface samples, high contact surfaces areas such as door handles, bathrooms, faucet handles, computer keyboards, cell phones, stairway rails, elevator buttons, and the like.
  • biological samples include tissue, blood, serum, plasma, saliva, nasopharyngeal swab or secretion, urine, cerebrospinal fluid, lymphatic fluid, sputum, cell lysate and the like.
  • An exemplary method can include detecting and/or quantifying a nucleic acid of interest.
  • the nucleic acid can be incubated with a composition including any of the microspheres and dsNA probes described herein for a suitable amount of time and under suitable conditions for the nucleic acid to displace the soluble strand of the dsNA probe such that the colorimetric indicator is no longer quenched.
  • the colorimetric indicator is measured by a suitable method, e.g., flow cytometry, FRET, spectrophotometry, etc., depending on the colorimetric indicator.
  • the amount of fluorescence or other indicator enables the detection and optionally quantification of the nucleic acid of interest.
  • the nucleic acid can be isolated from a sample as described herein using appropriate techniques known in the art.
  • Another exemplary method can include detecting and/or quantifying a nucleic acid of interest in a biological sample from a subject.
  • the biological sample can be taken from the subject and the nucleic acid can be isolated from the biological sample using appropriate techniques known in the art.
  • the isolated nucleic acid can then be incubated with a composition including any of the microspheres and dsNA probes described herein for a suitable amount of time and under suitable conditions for the nucleic acid to displace the soluble strand of the dsNA probe such that the colorimetric indicator is no longer quenched.
  • the colorimetric indicator is measured by a suitable method, e.g., flow cytometry, FRET, spectrophotometry, etc., depending on the colorimetric indicator.
  • a suitable method e.g., flow cytometry, FRET, spectrophotometry, etc.
  • the amount of fluorescence or other indicator enables the detection and optionally quantification of the nucleic acid of interest.
  • FIG. 1 illustrates displacement of the other strand 132 when a single stranded target NA molecule 142 hybridizes with the toehold region 128 of bound strand 122.
  • toehold domain 128 of the dsNA molecule can be designed to specifically match a single stranded NA molecule of a pathogen of interest.
  • a sample from a subject that includes such single stranded NA molecule would hybridize with bound strand 122 of composition 100 and displace the quencher of the colorimetric indicator 126 such that the colorimetric indicator is active (e.g., provide fluorescent signal).
  • strand 132 (the strand not bound to microsphere 110) can be soluble such that upon displacement of the strand 132 from bound strand 122, strand 132 is dissolved into the surroundings and unable to compete with the single stranded target NA molecule 142.
  • Strand 132 is referred to herein as the “soluble strand”.
  • FIG. 2 illustrates no displacement of the soluble strand 132 when an imperfectly mismatched single stranded NA molecule 144 is introduced to composition 100 and fails to displace soluble strand 132 from bound strand 122, as indicated on the plot to the right.
  • a sample that does not contain any single stranded NA molecule of a pathogen of interest would generate no signal.
  • composition 100 may include two or more different dsNA sequences targeting a variety of pathogens (e.g., variants of a pathogen) such that when any of the single stranded NA molecules of a pathogen of interest are introduced to composition 100, one or more soluble strands 132 may be displaced by the target NA molecule 142, indicating the presence of one or more variants of the pathogen of interest.
  • pathogens e.g., variants of a pathogen
  • composition 100 can further include a mismatched base pair 135, shown more clearly in FIGs. 2 and 3.
  • Mismatched base pair 135 between bound strand 122 and soluble strand 132 can be positioned in the recognition domain 124 of dsNA molecule 120.
  • mismatched base pair 135 may be positioned in the toehold domain 128 of bound strand 122.
  • mismatched base pair 135 may be positioned in both the recognition domain 124 and the toehold domain 128.
  • the bound strand 122 of the dsNA molecule 120 can include a COVID-19-specific nucleic acid.
  • the bound strand 122 can encode a COVID-19-specific protein.
  • the bound strand can include a sequence selected from the group provided in Table 1.
  • composition 300 can be quantitatively determined by measuring the extent of displacement activity in a soluble strand 332.
  • FIG. 3 provides a schematic of such quantitative assessments.
  • composition 300 can be initially fluorescent primary dsNA 320 with an unlabeled strand 322 immobilized on a microsphere 310 or particle as described supra.
  • the unlabeled strand 322 can be hybridized to FAM-labeled 15m hybridization strand 332 that can be designed to be selectively susceptible to displacement by an unlabeled target 342.
  • composition 300 Upon displacement of the hybridization strand 332, composition 300 is capable of forming nonfluorescent secondary duplexes 400.
  • primary dsNA 320 may include a recognition domain 324 and a toehold domain 328 on the immobilized unlabeled strand 322.
  • composition 300 may include a fluorescent tag on the soluble/hybridization strand 332 that is not quenchable.
  • composition 300 may further lack a quencher on the immobilized strand 322.
  • an unlabeled target 342 such as a pathogen RNA (e.g., such as SARS-CoV-2 RNA)
  • the dsNA 320 undergoes a disassociation of the fluorescent soluble strand 332 such that the composition 300 is no longer providing a signal that can be correlated into a quantitative assessment of the displacement activity.
  • the displacement activity of composition 300 may be adjusted by adding one or more mismatched base pairs 335 between the immobilized strand 322 and the fluorescent soluble strand 332.
  • plots of displacement activity for example compositions 100 are provided with LNA or DNA dsNA molecules 120 of varying length and number of mismatches.
  • LNA or DNA dsNA molecules 120 are hybridized with perfectly- matched sequence pairs (e.g., “LNA XQ” or “DNA XQ”), the target sequence is unable to compete and little to no fluorescence is produced unless the length of the LNA or DNA dsNA molecule is very short (e.g., 9 bases long).
  • LNA or DNA dsNA molecules 120 having a central abasic site exhibited fluorescence signals when introduced to a sample including a target single stranded NA molecule 142 (e.g., SARS-CoV-2 RNA). Although with a short sequence such as 9 bases or less, the soluble strand 132 may be displaced even without any target single stranded NA molecules.
  • preferred LNA dsNA molecules 120 present fluorescent signals only after the addition of a target of interest, such as SARS-CoV-2 RNA. As shown, 15nQ presents a desirable balance of stability and responsiveness due to neighboring 6 nucleotide toehold for 24 base-long target with a central abasic site.
  • FIG. 6 provides quantitative data of displacement activity of composition 300.
  • an unlabeled targets 342 e.g., SARS-CoV-2 RNA
  • an unlabeled mismatched target 344 e.g., var_RNA or scr_RNA sequence
  • FIG. 7 is a flow-chart of a method 700 for making nucleic acid detection platforms 100.
  • Method 700 can include isolating 702 a nucleic acid from a biological sample.
  • Method 700 can also include incubating 704 a nucleic acid with a composition 100 that includes microspheres 110 and at least one double-stranded nucleic acid (dsNA) molecule 120.
  • dsNA double-stranded nucleic acid
  • One strand 122 of the dsNA molecule 120 can be bound, at the 3’ end of the strand, to an outer surface 112 of the microspheres 120.
  • the bound strand 122 of the dsNA molecule 120 can include a recognition domain 124.
  • the bound strand 122 can also contain a quenchable colorimetric indicator 126 at a 5’ end.
  • the other strand 132 of the dsNA molecule 120 can be soluble and contain a quencher of the colorimetric indicator 136 at a 3’ end.
  • the soluble strand 132 can be configured to be displaced by the nucleic acid 142, thereby displacing the quencher of the colorimetric indicator such that the colorimetric indicator is active.
  • Method 700 can further include measuring 706 the colorimetric indicator to detect and/or quantify the nucleic acid 142.
  • RNA sequences were purchased from Integrated DNA Technologies (Coralville, IA), with standard desalting.
  • each immobilized probe possessed a 6-carboxyfluorescein (FAM) moiety at the 5’ end.
  • FAM 6-carboxyfluorescein
  • Each hybridization partner possessed an Iowa Black® Fluorescence Quencher (Q) moiety on the 3’ end.
  • Stock oligonucleotides solutions were prepared and stored in TE pH 8.0 (Sigma Aldrich, St. Louis, MO) at 100 pM.
  • probes and their hybridization partners Prior to coupling to microspheres, probes and their hybridization partners were annealed by heating the solutions (2: 1 quencher-capped hybridization partner to FAM-labeled biotinylated probes) to 94 °C for 2 min, then slowly cooling to room temperature. For microsphere coupling, working suspensions of 1.05 pm dia.
  • streptavidin- coated microspheres (Bangs Laboratory, Fishers, IN) were prepared by diluting stock from 1% w/v to 0.1% w/v in wash buffer (20 mM Tris (Sigma Aldrich), 1 M NaCl (Sigma Aldrich), 1 mM EDTA (Promega, Madison, WI), 0.0005% Triton X-100 (Sigma Aldrich)), per Bangs Product Data Sheet 721. Prior to oligonucleotide coupling, microspheres were washed 3 times by centrifuging at 14,000g for 3 min, aspirating supernatant, and resuspending in wash buffer.
  • RNA stock was added to the washed and dsprobe-functionalized microspheres for final concentration of 10 pM RNA and agitated for 15 minutes at 22 °C, 800rpm on a thermomixer. Microspheres were not washed following RNA addition.
  • a 5 pL microsphere suspension aliquot was added to 1000 pL PBS (Gibco, Waltham, MA).
  • MESF equivalent soluble fluorochrome
  • Example candidate dsprobe systems is provided in Table 2 (below).
  • Each dsprobe includes a biotinylated, 24 base-long 6-carboxyfluorescein (FAM)-tagged probe (i.e., LNA N 1_FAM or N 1_FAM) that is perfectly complementary to the 24 base-long RNA target (i.e., SARS-CoV-2 RNA).
  • FAM biotinylated, 24 base-long 6-carboxyfluorescein
  • LNA N 1_FAM or N 1_FAM RNA target
  • probes can be incubated with 1 of 20 Iowa Black® Fluorescence Quencher (Q)-capped hybridization partners of varying lengths (i.e., 9 to 21 bases), chemical modifications (i.e., locked vs.
  • any LNA-DNA chimera will be referred to as an LNA sequence.
  • a single-stranded or initially unhybridized segment ranging from 4 to 15 bases in length in each dsprobe. This single-stranded segment is intended to serve as a toehold for SARS-CoV-2 RNA to initiate duplex formation.
  • Table 2 provides example sequences and nomenclature of LNA, DNA, and RNA oligonucleotides employed in which the superscript “L” indicates a locked nucleotide in select FAM-functionalized probe and quencher-capped hybridization sequences;
  • X abasic nucleotide in select quencher-capped hybridization partners;
  • B C, G, or T;
  • D A, G, or T;
  • H A, C, or T in a mixture of model RNA sequence variants to SARS-CoV-2 RNA.
  • Each dsprobe is comprised of a 5' FAM moiety on the immobilized sequence and a 3' quencher (Q) on its hybridization partner.
  • DNA probe sequence is identical to the probe sequence named 2019-nCoV_N 1 -P on the Centers for Disease Control website (cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html) accessed on February 11, 2022.
  • the unlabeled RNA target must fully displace the quencher-capped LNA or DNA hybridization partner.
  • a fluorescence signal from the now unquenched N1_FAM should ensue from a successful toehold-mediated displacement event.
  • var_RNA a heterogeneous mixture of -243 similar, yet imperfectly-matched sequences (i.e., var_RNA) is included (see Table 2).
  • var_RNA a heterogeneous mixture of -243 similar, yet imperfectly-matched sequences
  • one or more mismatches occur within each var RNA segment intended to first hybridize to the toehold segment of the immobilized probe.
  • the greater number of total base-pair matches in the recognition+toehold segments may still favor displacement activity of any shorter, quenchercapped hybridization partner.
  • the scrambled RNA sequence i.e., scr RNA
  • the probe possesses only a few Watson-Crick base-pair matches with the probe and is thus not anticipated to participate in toehold-mediated displacement events.
  • the first dsprobe candidates consisted of LNA N1 FAM and quencher-capped LNA hybridization partners of three different lengths: 21 bases (i.e., LNA 2 IQ), 15 bases (i.e., LNA 15Q), and 9 bases (i.e., LNA 9Q). As shown in FIG. 4, these three dsprobe systems all exhibited little to no fluorescence signal prior to SARS-CoV-2 RNA addition as well in the presence of any noncomplementary RNA.
  • DNA is a reportedly weaker hybridization partner compared to LNA
  • stability and responsiveness of LNA N1 FAM hybridized to quencher-capped 9, 11, 13, and 15 base-long DNA with a central abasic site were also examined.
  • the longest of these DNA hybridization partners i.e. 15mQ
  • Nl_FAM 15mQ
  • Nl_FAM 15mQ
  • members of the heterogeneous RNA mixture do not appear competitive as replacements for 15mQ despite each var RNA sequence having more total base-pair matches than 15mQ for N 1 FAM probe including a continuous 11 base-long match to the recognition segment and multiple, though noncontinuous base-pair matches for the remainder of the recognition segment and adjacent toehold segment as indicated in Table 3.
  • the dsprobe possesses a 5' FAM moiety on the immobilized sequence and a 3' quencher (Q) on its hybridization partner. To facilitate comparison to the quencher- capped hybridization partner in the dsprobe, all RNA sequences are shown 3'— >5'.
  • the duplex density is measured before RNA addition, then after adding scr RNA (to assess simple dissociation events) and var RNA (to assess displacement by imperfectly-matched RNA). As shown in FIG. 6, less than 4% decrease in duplex densities occurs with either of these RNA controls. In contrast, the primary duplex density decreases by 88% in the presence of SARS-CoV-2 RNA. Based on the overall stability of Nl: 15m_FAM in the presence of noncomplementary or imperfectly-matched RNA, this marked decrease in the presence of SARS-CoV-2 RNA must be due to displacement of the shorter, FAM-labelled sequence with a central abasic site as illustrated in FIG. 3 (right).
  • this platform can be further expanded to both capture and separate targets via FACS-based sorting (i.e., separate fluorescent from quenched probe-functionalized microspheres) for further analysis (e.g., sequencing) as warranted.
  • FACS-based sorting i.e., separate fluorescent from quenched probe-functionalized microspheres
  • sequencing e.g., sequencing
  • Example 3 Exemplary Isolation of Nucleic Acid from a Sample
  • a sample is obtained.
  • the sample include a biological sample, a water sample, a wastewater sample, an environmental sample, a food sample, a food contact surface sample, and/or a high surface contact area sample.
  • biological samples include tissue, blood, serum, plasma, saliva, nasopharyngeal swab or secretion, urine, cerebrospinal fluid, lymphatic fluid, sputum, cell lysate, and the like.
  • a nucleic acid is isolated from the sample using conventional nucleic acid isolating techniques.
  • the isolated nucleic acid is then incubated with a microsphere comprising a dsprobe according to any of the embodiments described herein under conditions suitable for the isolated nucleic acid to displace the soluble strand of the dsprobe.
  • the bound strand of the dsprobe is then able to fluoresce.
  • the fluorescence can be detected and/or quantitated by any conventional methods, such as for example and not limitation, a high- throughput spectrophotometer, a fluorescence activated cell sorting (FACS) system, flow cytometer, or a color activated cell sorting system.
  • FACS fluorescence activated cell sorting
  • a composition comprising:
  • dsNA double-stranded nucleic acid
  • the bound strand of the at least one dsNA molecule comprises a recognition domain and contains a quenchable colorimetric indicator at a 5’ end
  • the other strand of the at least one dsNA molecule is soluble and contains a quencher of the colorimetric indicator at a 3 ’ end.
  • composition of item 3, wherein the toehold domain comprises between
  • composition of any of items 1-8, wherein the dsNA molecule comprises
  • DNA DNA, RNA, or LNA.
  • composition of any of items 1-9, wherein the bound strand of the at least one dsNA molecule comprises a COVID- 19-specific nucleic acid, or encodes a COVID- 19-specific protein.
  • kits for detecting the presence of a nucleic acid in a sample comprising:
  • dsNA double-stranded nucleic acid
  • the bound strand of the at least one dsNA molecule comprises a recognition domain and contains a quenchable colorimetric indicator at a 5’ end
  • DNA DNA, RNA, or LNA.
  • kit of any of items 12-20, wherein the bound strand of the at least one dsNA molecule comprises a COVID- 19-specific nucleic acid, or encodes a COVID-19- specific protein.
  • the soluble strand of the at least one dsNA molecule has a sequence selected from the group consisting of SEQ ID NOs: 2-11, 13-20, and 22.
  • sample comprises a biological sample, a water sample, a wastewater sample, an environmental sample, a food sample, a food contact surface sample, and/or a high surface contact area sample.
  • kits of any of items 12-23, wherein the biological sample comprises tissue, blood, serum, plasma, saliva, nasopharyngeal swab or secretion, urine, cerebrospinal fluid, lymphatic fluid, sputum, cell lysate, and the like.
  • a method of detecting and/or quantifying a nucleic acid comprising:
  • dsNA double-stranded nucleic acid
  • the bound strand of the at least one dsNA molecule comprises a recognition domain and contains a quenchable colorimetric indicator at a 5’ end
  • the other strand of the at least one dsNA molecule is soluble and contains a quencher of the colorimetric indicator at a 3 ’ end
  • the soluble strand of the at least one dsNA molecule is configured to be displaced by the nucleic acid, thereby displacing the quencher of the colorimetric indicator such that the colorimetric indicator is active;
  • [00163] 26 The method of item 25, wherein the measuring step is performed by a high-throughput spectrophotometer, a fluorescence activated cell sorting (FACS) system, flow cytometer, or a color activated cell sorting system.
  • FACS fluorescence activated cell sorting
  • DNA DNA, RNA, or LNA.
  • [00170] 33 The method of any of items 25-32, wherein the bound strand of the at least one dsNA molecule comprises a COVID-19-specific nucleic acid, or encodes a COVID-19- specific protein.
  • [00173] 35 A method of detecting a nucleic acid in a biological sample from a patient, the method comprising:
  • dsNA double-stranded nucleic acid
  • the bound strand of the at least one dsNA molecule comprises a recognition domain and contains a quenchable colorimetric indicator at a 5’ end
  • the soluble strand of the at least one dsNA molecule is configured to be displaced by the nucleic acid, thereby displacing the quencher of the colorimetric indicator such that the colorimetric indicator is active;
  • DNA DNA, RNA, or LNA.
  • the soluble strand of the at least one dsNA molecule has a sequence selected from the group consisting of SEQ ID NOs: 2-11, 13-20, and 22.

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Abstract

Un mode de réalisation de la présente divulgation donné à titre d'exemple concerne une composition comprenant des microsphères et au moins une molécule d'acide nucléique double brin (ANdb). Un brin de le ladite au moins une molécule d'ANdb est lié à une surface externe des microsphères à une extrémité 3'. Le brin lié de ladite au moins une molécule d'ANdb comprend un domaine de reconnaissance et contient un indicateur colorimétrique désactivable à une extrémité 5'. L'autre brin de ladite au moins une molécule d'ANdb est soluble et contient un désactiveur de l'indicateur colorimétrique à une extrémité 3'. La présente divulgation concerne également des procédés de fabrication d'une composition comprenant des microsphères et au moins une molécule d'acide nucléique double brin (ANdb).
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